This is a proposal to investigate the control of cell-type-specific gene expression in differentiated cells. Using highly differentiated rat hepatoma cells growing in vitro as a model system, we will apply an integrated genetic/molecular biological approach to study the regulation of expression of liver-spedific genes. Genetic analyses will be performed with the use of a novel monochromosomal hybrid panel (MHP). This panel will consist of 20 rat hepatoma hybrid clones, each containing a single, specific mouse fibroblast chromosome. The MHP will be used (a) to map the structural genes encoding a number of liver-specific traits, and (b) to define fibroblast genes which cause extinction of hepatic gene expression in intertypic hybrid cells. Liver-specific cDNA clones will be used to determine whether extinction of hepatic traits reflects gene regulation at the level of transcription. The recombiant molecules will also be used to probe the structural organization of liver-specific genes in cells which differ in their expression of those sequences. Liver-specific alcohol dehydrogenase (ADH) will be studied in detail. We propose (a) to develop selective systems for and against this trait, (b) to isolate somatic cell mutants with altered ADH expression, (c) to isolate cDNA clones encoding ADH, and (d) to perform gene transfer studies in which ADH structural or regulatory sequences are introduced into various cell types. The proposed studies should contribute to our understanding of the genetic and molecular mechanisms which regulate gene expression in differentiated cells. This will be important for designing approaches to study perturbations of these regulatory controls which may underlie abnormal developmental situations and neoplasia.